Thymidylate synthase (TS) Is the enzyme that catalyzes the de novo biosynthesis of thymidylic acid. The enzyme Is essential in proliferating cells and is an important target enzyme In cancer chemotherapy. The overall goal of this project Is to understand the biochemical mechanisms for controlling the expression of the mouse TS gene In growth-stimulated cells. The studies In this proposal will focus on the Identification of the regulatory elements and trans-acting factors and explore In more detail the complex Interactions that are Important for proper regulation of this Important gene. Three specific aims are described. The first aim is to determine the locations of regulatory sequences that are upstream of the essential promoter elements. The trans-acting factors that Interact with the regulatory sequences will be studied. Qualitative or quantitative changes In these factors will be measured M cells progress from G1 through S phase. The possible relationship between these factors and the E2F transcription factor, or the RB tumor suppressor protein will be Investigated. If novel trans-acting factors are Identified, they will be characterized In detail. The second aim is to determine why Introns are necessary for proper regulation of TS minigenes. The ability of Introns from genes that are not cell cycle-regulated to restore proper regulation to an intronless TS minigene will be examined. The redundant (and perhaps autonomous) regulatory sequences that are located within Intron 2 will be Identified, and the mechanism by which they bring about growth- regulated expression will be examined. The third aim is to study in more detail the bidirectional nature of the mouse TS promoter. A preliminary characterization of the opposite direction promoter and the transcripts It may encode will be performed. The regulation of the opposite direction gene will be studied in growth-stimulated cells to determine if It is controlled In the same manner as the TS gene. If the 5' flanking sequences are found to block bidirectional transcription, the mechanism responsible for this inhibition will be analyzed. The proposed studies will increase our understanding of the mechanisms by which- cells regulate the progression from G1 to S phase. They will also provide additional-insight into the mechanisms by 'which sequences in the 5' flanking region of the gene as well as Introns affect gene expression.